Production of liver extract



25 A further object is to provide an improved ties of thephysiologically inactive ingredients 26 ording tothis patent, liver ismacerated and to produce a solution containingthe desired Patented it...23, 1936 2,045,266

UNITED STATES PATENT OFFICE PRODUCTION OF LIVER EXTRACT Frederic Fenger,Chicago, Ill., asslgnor to Armour 2.111151 gompany, Chicago, 111., acorporation of No Drawing. Application April 80, rest, Serial No.323,175

3 Claims. (oi. rev-r4) The p n invention relates to mammfilian subjectedto extraction with an aqueous-alcoe t and has particular reference toholic solution from which is Subsequently preprovements in theproduction of extracts, such cipitated by strong alcohol aphysiologically a e as liver extra a kidney extract, h v p ytivefraction employed. i'or treating secondary 5 siologically activeprinciples for internal admina ia 5 istration in augmentation of naturalbody pro- The constituents of an aqueous or a weak alduction. coholicextract of liver are extremely compli- It has long been recognized thatwhere the cated and not entirely understood. Included in animal systemis deficient in some active printhese constituents are physiologicallyactive prin- Li ciple to such an extent as to cause depletion of cipleswhich vary in their solubilities in alcohol.

the physical system, it is possible to restore con- For example, alcoholsolutions of increasing ditions approaching normal, at least for alimited strength will cause the selective precipitation of timeSubsequent to treatment. y dministerin these physiologically activeprinciples. The acaconcentrated extract of the deficient physiologitiveingredients soluble in alcoholic solutions in cally active principleobtained from some other 50 to 70 per cent strength are used extensively15 animal. ,Principally, the extracts are adminisin the treatment ofsecondary anemia, while the tered orally, but in many cases the extractsare fraction soluble in alcoholic solutions from 70 to supplied to theanimal system hypodermatically. 99 per cent have been used moreextensively in A principal object of the present invention is to thetreatment of pernicious anemia.

provide an improved process for producing mam- Along with thephysiologically active ingredi- 20 malian extracts. cuts of the extractare included many complex An additional object is the provision of anproteins and otherconstituents apparently of a improved liver extractand process of making carbohydrate nature. The extracts which have thesame. been known heretofore include copious quantiprocess for producingliver extract having an which'increase the total solids content of theexunusually high concentration of physiologically tract considerablywithout adding-to its efficacy. active ingredients, an unusually lowconcentra- In fact, particularly where the extract is to be tion ofphysiologically inactive solids, and having used intravenously orhypodermatically, the use amore acceptable taste and odor than extractsI of liver extract has caused considerable discom- 30 of this type knownheretofore. fiture in a patient. In the case of certain liver These andother objects will be evident from a extracts now on the market, thereis a total solids consideration of the following description of acontent of 30-per cent or more, andonly a small preferred embodiment ofmy invention. 1 portionof this extract consists of physiologically Thegeneral process of producing mammalian active ingredients. Therefore, it.will be seen extracts consists in macerating the body portion that apatient is required to consume a relatively from which it is desired toobtain physiologically large quantity of solids, some of which aredetriactive constituents and soaking theimaterial to mental, in order tomake up the bodily deficiency produce an extract which-subsequently isconin physiologically active ingredients. 40 centrated to the desiredextent. Solvents, such The present invention is based upon my discovasalcohol, are employed variously in the producery that a greatly improvedextract can be obtion and purification, of the extract. Probably tainedby producing an aqueous solution of the the principally used animalextract is liver exactive ingredients and subjecting this aqueous tract,and it is to the preparation of this ma- 'solutionto'ye'ast fermentationin order to conterial' that the present invention is particularly vertthe carbohydrate constituent of the extract adapted. For this. reasonthefollowing descrip into alcoholic form. Furthermore, the fermentedtionwill be" made with. reference to liver exextract is subjected to adigestion process by tract, although it vwill'be understood that othertreatment with an acid-pepsin, thereby eliminatextr'acts, such as kidneyand tissue extracts-also ing certain undesirable proteins and renderingmay be produced toadvantage'in accordance the extract considerablymoredesirable from the Q with my'processy v v J physiological standpoint;After this treatment A process for producing liverlextracts which theextract can be treated with alcohol topre-v has met withconsiderablefavor is described in cipitate still further ingredients. By properco'n-' Patent No; 1,813,788, issued-July '7, 1931. Actrol of the amountof alcohol used, it is possible physiologically active ingredients withthe e'xclu sionof the 'precipitated'materials.

The process is carried out by macerating a quantity of liversfreshly'removed from the animal and preferably still containing thebodily heat and contacting the macerated tissue with water for a lengthof time sufficient to extract the desired physiologically activeingredients of the liver. By heating the extract to a temperature of 85C. the extraction of the active constituents is facilitated and certainheat coagulable proteins are precipitated. Subsequently, the extract iscondensed at reduced pressure and at a low temperature, such as 37.5 C.,or blood heat, until the solids content of the extract reaches aconcentration suitable for yeast fermentation. As will be understood,the optimum solids concentration will vary with the different tissuesemployed. After being cooled under aseptic conditions, the concentratedextract is inoculated with a pure culture of yeast, and is set aside,well covered and protected from contamination, at a temperature suitablefor optimum alcoholic fermentation. In some cases organisms or sugarfermenting enzymes, other than yeast, will be found desirable forconverting the carbohydrate constituents of the extract into alcoholicform but in general I have found it preferable to employ yeastfermentation.

'When a test with Fehlings solution or other carbohydrate detectingmaterial shows that the sugar present in the extract has been convertedinto alcohol, the fermentation is stopped and the fermented concentrateis then subjected to a digestion step which causes hydrolysis of theyeast and/or other sugar fermenting organisms and enzymes introducedinto the concentrate or formed during the fermentation process. Thedigestion step also destroys undesirable yeast and/or other metabolates.This digestion step preferably is carried out by adding to thefermentated concentrate a mixture of hydrochloric acid and pepsin. Otheracids, such as sulphuric, phosphoric, tartaric, etc. may be employed inplace of or in addition to hydrochloric acid.

After the digestion has gone to the desired extent, the concentrate istreated with a sumcient quantity of dilute alkali to readjust thehydrogen ion concentration or pH of the concentrate to approximately thepH of the original extract.

Thereafter a sumcient quantity of alcohol is added to the concentrate tobring the alcoholic concentration up to between and per cent. Other wellknown solvents of the physiologically active constituents of liver maybe employed at this point in place of, or in addition to, the alcohol.For instance, ethyl alcohol, or a mixture of ethyl and methyl alcoholmay be employed.

The alcoholic treatment causes the precipitation of certain proteins andafter proper settling to remove these precipitating inert proteins andother unsoluble constituents the non-alcoholic liquid is poured off orotherwise. separated and condensed at a relatively low temperature andunder reduced pressure to the desired solids con- I tent. Ordinarily,the evaporation will continue until the desired material is in a drypaste, or concentrated liquid form. In any of these forms the liverextract is ready for administration. Where the material is to be usedhypodermatically, it may be found desirable to make a still furtherpurification.

As a specificv example of the process as described hereinbefore andembodying my invention, the following details of a particular run aremacerated with 50 gallons of water.

- given. Itwill'be understood that this example is merely one of manymodifications of the process, and that the proportions of materialsemployed and the manner of treatment will vary in accordance with theparticular material used and the result which it is desired to produce.7

A quantity of healthy, fresh mammalian livers still containing theanimal heat, and weighing approximately 150 pounds, are finely mincedand The mix- 10 ture is warmed sufllciently to extract thephysiologically active constituents and to coagulate certain proteinswhich otherwise would remain in the solution. The aqueous extract isstrained or filtered from the insoluble constituents and the 15 residueis extracted with an additional quantity of water, the two liquidextracts being combined. The combined extracts are evaporated underreduced pressure and at a temperature of approximately 375 C. to asolids content of between 25 and 30 per cent.

After cooling the concentrate under aseptic conditions to a temperatureapproximately 75 F. the concentrate is inoculated with two fluid ouncesof freshly prepared, pure and active culture of yeast. The vessel inwhich the inoculated concentrate is contained is covered over to preventcontamination. Fermentation is allowed to proceed until the concentrategives a negative test to Fehlings solution. During the fermentationprocess carbon dioxide is produced and forms a protecting blanket overthe top of the concentrate, thereby preventing the oxidation whichotherwise is liable to occur.

After the fermentation process has gone to to completion, the fermentingextract is subjected to a digestion step in which 25 c. c. ofconcentrated hydrochloric acid, diluted with c. c. of water, and 10grams of pepsin are added to the concentrate for each gallon thereof.The concentrate is then maintained at a temperature of approximately F.for two hours, the mixture then being allowed to cool slowly to roomtemperature. Preferably the cooling extends over night. I

Dilute sodium hydroxide solution is added to the digested concentrateuntil the reaction of the concentrate reaches a hydrogen ionconcentration of from 5.0 to 5.5 on the pH scale, this being the normalhydrogen ion concentration of the original, fresh aqueous extract fromthe mammalian livers. Any suitable alkaline material, such as sodiumhydroxide, potassium hydroxide or sodium carbonate, may be employed forthis neutralization step. 55

A sumcient quantity of alcohol is then added to the concentrate to bringthe alcoholic concentration to 85 per cent. An alcoholic solutioncontaining 10 parts of methyl alcohol and 100 parts of ethyl alcohol maybe used, if desired, since 60 this denatured mixture is considered moreeconomical than pure alcohol.

The mixture then is set aside at a temperature of approximately 40 F.,whereupon physiologically inert proteins and other insolubleconstituents are precipitated from the extract. This cold alcoholfractionation precipitates other materials, such as phosphates, butleaves the desired physiologically active constituents in solution. Aconsiderably sharper fraction is obtained where the mixture previouslyhas been subjected to yeast fermentation than has been obtainedheretofore. The clear supernatant alcoholic to maintain the water.content of the extract sumciently high to permit adequate yeast growth.Ordinarily it is preferredto employ an extract having not more than;about 10 per cent alcohol, 3 and in most cases an even lower percentageof alcohol is preferred.- a

Liver extracts known heretofore have a characteristic taste and odorwhich makes their use for oral administration undesirable. Frequently.the liver odor and taste are so strong as to make the material quiterepulsive to the patient. An important feature of my improved processisthe discovery that it removes this undesirable taste and odor andleaves a product not at all offensive to the patient. The nature of thereactionby which the taste and odor are removed is not brown. The factremains, however, that some action of theyeast upon the extract changesthe odor and taste-imparting ingredients to such an extent as to destroytheir normal characteristics, thereby leaving a sweet product. As theundesirable odor and taste-imparting constituents of liver extract seemto interfere with the digestive system of the patient, possibly throughthe nausea caused by their reaction on the sense organs, it will be seenthat my process will act in this further manner to increase the efficacyof and, the final extract containing the erythropi-.

etic principles and/or other physiologically active substances of value,such as vitamins B1 and B2, used in the treatment of nutritionaldisorders,

. such as anemia, beriberi, and pellagra, have not Particularly in thecase of liver extractused for hypodermatic administration, thiscarbohydrate content is objectionable. I

I have found that although the yeast fermentation converts thecarbohydrates quantitatively into alcohol, which may be distilled fromthe extract, the physiologically active ingredients are notdeleteriously affected. The sugars normally present in liver extractseem to exert a buffer action which is eliminated by the yeastfermentation. This elimination and the acid-pepsin hy-' drolysis towhich the extract is subjected after the yeast fermentation conditionsthe extract so that the subsequent alcohol separation of thephysiologically active fraction from the inert material becomes sharperand more clear cut.

' Aqueous extracts from mammalian tissuecontain certain colloidalprotein substances of high viscosity which are not precipitated by heat.These colloids are commonly known as gelatins".

chanically, or adsorbed, considerable quantities 3 When the aqueousextract of the physiologically active constituents and these colloidsare condensed, the latter have a tendency to gelatinize the concentrate,particularly when chilled. This 7 tendency isobiectionable. since thealcohol frac- 5 tionation ofthe solution is carried out at a lowtemperature such 40 1''. .when the alcohol is added to the solutionthese gelatinous colloids are precipitated. carrying down with them me-'of the physiologically active substances- Separation of thesephysiologically active substances from the precipitate by washing withdilute al- 1 cohoi is practically impossible. e I have found that theacid-pepsin hydrolysis 15 alcohol fractionation of the physiologicallyactive 20 constituents becomes sharp and clear cut, a maxi-' mum ofphysiologically active substances remaining in the solution. The smallamounts of physiologically active substances which are carried downwiththe precipitate upon the alcohol 25 fractionation may be recovered bywashing with dilutealcohol.

It will be seen that the process is susceptible of regulation to producethe desired type and con-- centration of active constituents, and inevery case 30 there is produced a product which issuperior in P i y.taste, odor and eillcacy to th pmducts known heretofore. In some cases,11; in um J cient to omit the final step of alcohol precipitation 1 bedispensed with. However, it is preferred to;

and in other cases the acid-pepsin di estion may I employ both of thesesteps, since, the resultant product is more desirable for most purposesrIn any case the conversion of the carbohydrate'frac- 1 tion of theconstituents into alcohol results'in a palatable product which extensiveuse has proven" to besuperior to the productsknown heretofore It will beunderstood that the foregoing description is for the purpose ofillustration'and explanation and that various changes in the procedureoutlined are possible. All such changes and modifications are intendedto be included in the appended claims. I

i claim: v 1. The process of preparing a concentrated extract ofmammalian tissue which comprises producing an extract of the tissuecontaining a sum-- cient quantityof water to permit alcoholic fer-mentation, subjecting the extract to alcoholic fermentation to removefermentable carbohydrates therefrom, and concentratingthe fermentedextract to the desired extent. a

g 2. The process of preparing a vconcentrated extract of mammaliantissue which comprises producing an extract of the tissuecontaining a 0sufllcient quantity of water to permit alcoholic fermentation.subjecting the extract to alcoholic fermentation to remove fermentablecarbohydrates therefrom, subjecting the fermented extract to a digestivestep, and concentrating the 5 sumciently large volume of alcohol toprecipitate a portion only of the dissolved substances in said extract,separatingthe remaining alcoholic solution, and concentrating saidsolution by evaporaextract of liver which comprises producing an aqueousextract of a quantity of liver, concentratingsaid-extract, subjectingthe extract to yeast Jermentation, subjecting the fermented extract to-adigesting step. adding-a sumcientv quantity I ofalcohol to theextract toprecipitate a portion only of thedissolved substances ins-the extract,

.and concentrating said extract desiredex 'tent 6. The process ofproducing aphysiologically active extract of liver whichfcomprisesproducing an aqueous ,extractof a quantity of -livenconverting "thecarbohydrates in-saidextract into alcohcl, and concentrating the extractto the desired extent.

7; The process of preparing a concentrated mammalian tissue extractwhich comprises macerating thetissue, subjecting the macerated tis- .sueto extraction with water, applying sumcient but during the extractionstep to cause the active constituents of the tissue to dissolve and toprecipitate heat coagulahle protein, condensing the extract under vacuumand at a relatively low temperature until the extract has a solidscontent suitable for yeast fermentation, inoculating theconcentratedextract withyeast, subjecting the inoculated extract to alcoholicfermentation to remove fermentable carbohydrates there- ;from,subjecting the fermented extract to treatment with hydrochloric acid andpepsin, adjusting the pH of the extract to substantially that of theoriginal extract, adding a sufficient quantity of alcohol to make ,thealcoholic concentration of the extract between and per cent, settlingthe extract, removing the clear alcoholic fraction thereof, andconcentrating said fraction --by evaporation under vacuum.

' 8. The-process of producing a concentrated extract from kidney tissue,which comprises producing an aqueous extract of said tissue, removingwater from said extract to produce a concentrate, subjecting saidconcentrate to alcoholic fermentation to remove carbohydrates therefrom,

V subjecting the fermentedconcentrate to a digestive step, and furtherconcentrating the product of said digestion.

FREDERIC FENGER.

